Atypical E3 ligase HOIL1 promotes MDA5 signaling and ester-linked ubiquitination of LGP2

The RIG-I-like receptors, RIG-I and MDA5, are innate sensors of RNA virus infections that are critical for mounting a robust antiviral immune response. Heme-oxidized IRP2 ligase 1 (HOIL1), a component of the linear ubiquitin chain assembly complex (LUBAC), is essential for interferon induction in response to viruses sensed by MDA5, but not viruses sensed by RIG-I; however, the underlying mechanism remains unclear. LUBAC contains two atypical E3 ubiquitin ligases, HOIL1 and HOIP. While HOIP generates "linear" polyubiquitin chains, HOIL1 catalyzes ester-linked ubiquitination of serine and threonine residues and glucosaccharides. Here, we show that HOIL1 E3 ligase activity is critical for MDA5-dependent interferon induction, whereas HOIP E3 ligase activity plays a minor role. Mechanistically, HOIL1 E3 ligase activity was required for MDA5 translocation to mitochondrion-associated membranes and the formation of MAVS aggregates. We further identified LGP2, a positive regulator of MDA5 oligomerization, as a HOIL1-interacting protein and substrate for ester-linked ubiquitination. In mice, loss of HOIL1 or its E3 ligase activity markedly impaired early interferon responses following infection with viruses sensed by MDA5. While HOIL1 deficiency resulted in increased viral burden and reduced survival, loss of HOIL1 ligase activity alone did not, indicating that HOIL1 exerts additional functions during antiviral immunity. Collectively, these findings establish ester-linked ubiquitination by HOIL1 as a critical mechanism regulating MDA5 signaling.IMPORTANCEThe rapid and robust production of interferon is critical for controlling viral infections. Cytoplasmic RNA sensors, RIG-I and MDA5, detect infection with distinct groups of viruses, leading to the transcriptional induction of interferon. However, inappropriate production of interferon triggered by aberrant MDA5 activation by cellular RNAs causes severe inflammatory disorders. Here, we report that the ubiquitin ligase activity of heme-oxidized IRP2 ligase 1 (HOIL1) is critical for the production of interferon in response to viruses that are sensed by MDA5 in cells and mice. We identify the third RIG-I-like receptor, LGP2, as a substrate for ester-linked ubiquitination by HOIL1, and show that, like LGP2, HOIL1 ubiquitin ligase activity is required for the efficient oligomerization of MDA5 and the initiation of downstream signaling leading to the induction of interferon. These findings reveal a new regulator of MDA5-driven interferon production and identify a potential therapeutic target for MDA5-mediated inflammatory diseases.